Four major approaches have been taken to define non-classical multidrug resistance in cancer. In the first, we isolated KB cells (a subclone of HeLa) resistant to increasing levels of cisplatin and demonstrated multidrug resistance to arsenite and cadmium, to methotrexate, and to nucleoside analogs. This cross-resistance pattern is due to reduced uptake of each of these agents because their receptors have been relocalized from the cell surface into the cytoplasm of the cell. This relocalization of surface transporters appears to be due to altered recycling of these transporters due to alterations in endocytic recycling compartments in cisplatin-resistant cells. One consequence of reduced cell surface transporters is a reduction in glucose uptake and altered mitochondrial metabolism mediated by SIRT1. To determine the molecular basis of this pleiotrophic defect in cisplatin-resistant cells, we created a cDNA library from resistant cells and transfected it into sensitive cells to determine which genes confer multidrug resistance including resistance to cisplatin. Several cDNAs, including those encoding metallotheinein, heat shock proteins, ribosomal proteins, a selenoprotein, and membrane proteins were isolated and their ability to confer multidrug-resistance is being determined. The mitochondrial transporter ABCB6 has also been associated with resistance to arsenite and cisplatin. ABCB6 is expressed in the outer mitochondrial membrane and plasma membrane of resistant cells, but its precise function remains to be determined. A second approach was to evaluate the unique features of melanoma cells that contribute to multidrug-resistance. One obvious feature of melanoma cells is the melanosome, a lysosome-derived organelle in which pigment formation takes place. We have shown that cisplatin is sequestered in this organelle, independent of extent of melanin formation, and extruded with melanosomes into the medium, reducing nuclear accumulation of this anti-cancer drug. Studies are underway to determine whether ABCB5, a transporter expressed at high levels in pigmented cells such as melanocytes and melanomas, contributes to the melanosomal sequestration seen in melanomas. A third approach is to determine the molecular basis of multidrug resistance in cancer stem cells. As part of an NIH Breast Cancer Consortium, supported in part by breast cancer stamp funds, we have begun to isolate breast cancer stem cells and normal breast epithelial stem cells from surgical samples. CD133 positive cells from these cell populations can be propagated in tissue culture using approaches previously developed for growing human ES cells. Our goal is to evaluate the expression of multidrug-resistance genes, including both classical ABC efflux transporters and uptake transporters, as well as non-classical mechanisms of multidrug resistance, in cancer stem cells derived from these surgical speciments. Finally, we have developed a Taqman Low Density Array (TLDA) microfluidic chip to detect mRNA expression of 380 different putative drug resistance genes and demonstrated that it is a sensitive, accurate, reproducible, and robust way to measure mRNA levels in tumor samples. Previous work from our laboratory indicates that mRNA measurements of levels of drug-resistance genes are, to a first approximation, predictive of functional expression of drug-resistance mechanisms. This drug-resistance chip is being applied to analysis of human cancers that show either response or lack of response to specific chemotherapy. We have initiated our studies on ovarian cancer, where cancers frequently respond to chemotherapy and then become resistant.